FIND is a global non-profit driving diagnostic innovation ... · 8 labs used this protocol in 2012...

Overview of diagnostic tools to advance clinical trials

(confirmatory diagnosis, assays for vaccine development)

Devy Emperador – Scientific Officer, Pandemic Preparedness

[email protected]

WHO consultation on RVFV therapeutics and vaccine evaluation - WHO R&D Blueprint Meeting

1st Nov 2019; Geneva, Switzerland

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FIND is a global non-profit driving diagnostic innovation to combat major

diseases affecting the world’s poorest populations

WHO Collaborating Centre for Laboratory Strengthening

& Diagnostic Technology Evaluation

WHO SAGE-IVD member

ISO-certified quality management system for IVD clinical trials

We address market failure by partnering

to develop and deliver diagnostic solutions

to LMICs

ANTIMICROBIAL

RESISTANCE

HEPATITIS C

& HIVMALARIA & FEVER

NEGLECTED

TROPICAL DISEASES

PANDEMIC

PREPAREDNESSTUBERCULOSIS

Geneva

(HQ)

South

Africa

India

Viet Nam

Kenya

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ANTIMICROBIAL RESISTANCE

Without action, 10 million deaths every year

predicted by 2050

MALARIA & FEVER

In Africa alone, over 600 million childhood fevers occur

every year – which may or may not be due to malaria

HEPATITIS C & HIV

71 million people affected by HCV but only

1 in 5 know their status

NEGLECTED TROPICAL DISEASES

Affect more than 1 billion people, yet suffer from

historical lack of attention

PANDEMIC PREPAREDNESS

6 out of 10 WHO “Blueprint” pathogens have significant

unmet diagnostic needs

TUBERCULOSIS

3.6 million cases missed in 2017 and the

majority of infected children go undiagnosed

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In the 2014–16 Ebola

outbreak, a point-of-care

diagnostic could have saved:

70% of

lives lost

80% of

all costs

Diagnostics save lives, and money

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What happens without diagnostics?

Impact of not closing the diagnostic gap

– Delayed identification of outbreaks leading to escalation and spread

– Increased costs of local, national, international response

– Delays in vaccine and therapeutic evaluations

– Limited understanding of epidemiology of disease

Clinical trials for vaccines and therapeutics require well characterized

diagnostics:

– Clear planning for trials based on sound epidemiological data

– Clear enrollment criteria for patients based on definitive diagnosis

– Early identification of at risk populations for tailored clinical trials

Delays in addressing the diagnostic gaps will cause delays in initiation of

clinical trials, increased costs and potentially inappropriate site selection

PANDEMIC PREPAREDNESS

6 out of 10 WHO “Blueprint” pathogens have significant

unmet diagnostic needs

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Diagnostics for Rift Valley Fever

Pathogen ID

– Virus isolation

– RT-PCR

– Antigen detection (ELISA, immunohistochemistry)

Serological tests

– Immunofluorescence assays

– Virus neutralization

– ELISA

Biocontainment needs: High (BSL-3)

Current testing location: Limited to reference laboratories

Source: Hartman A. (2017). Rift Valley Fever. Clinics in laboratory medicine, 37(2), 285–301. doi:10.1016/j.cll.2017.01.004. Mansfield KL et al. (2015). Rift Valley fever virus: A review of diagnosis and vaccination, and implications for

emergence in Europe. Vaccine, 33(42), 5520-5531. https://doi.org/10.1016/j.vaccine.2015.08.020. Paweska JT. (2015). Rift Valley fever. Revue scientifique et technique, 34, 375-389.

Figure: Biomarker dynamics in Sheep Experimentally Infected with Natural RVFV

Note: Viremic period can often be very short, as little as 2 or 3 days.

https://doi.org/10.1016/j.vaccine.2015.08.020

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Status of commercial assays for Rift Valley Fever

Type Product name Species Company Regulatory Comments

NAT RealStar RVFV RT-PCR Human Altona CE No published independent studies

NAT RVFV Human TIB MolBiol RUO No published independent studies

NAT Rift Valley Virus (RVFV) Real Time RT-PCR

Reagent

Human LifeRiver CE No published independent studies

NAT FTD RVFV Human Fast-Track Diagnostics RUO No published independent studies

NAT Rift Valley Fever Virus RT-PCR kit; EBOV+RVFV

and EBOV+RVFV+YFV PCR kits

Human Genekam Biotech Ag RUO No published independent studies

NAT Rift Valley Fever virus Human PCRMax RUO No published indpendent studies

NAT QPCR Kit, RNA, Rift Valley Fever Virus Human Techne/Cole Palmer RUO No published independent studies

NAT

(in dev)

field-ready RT-PCR, assay for RVFV, "Lab-in-a-

suitcase"

- USDA and GeneReach not known Status unknown

PCR +

Luminex

(in dev)

platform for surveillance, serology, a panel of

diseases, such as RVFV and Blue Tongue

- USDA not known Status unknown

Source: Mansfield KL et al. (2015). Rift Valley fever virus: A review of diagnosis and vaccination, and implications for emergence in Europe. Vaccine, 33(42), 5520-5531. https://doi.org/10.1016/j.vaccine.2015.08.020. Rift Valley Fever

Diagnostics – FIND Landscape (2017).


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ELISA IgG ELISA assay ID Screen Rift Valley Fever

Competition (IgM + IgG)

Multi-species ID-vet Veterinary Good performance in 9 labs in 2015 animal

EQA study and animal ring trial

ELISA ID Screen Rift Valley Fever IgM Capture Ruminants ID-vet Veterinary Good performance in 10 labs in 2015 animal

EQA study

ELISA Ingezim FVR DR 13-FVR.K0 (IgM) Ruminants Ingenasa Veterinary No published independent studies identified

ELISA Ingezim FVR Compac 13.FVR.K Ruminants Ingenasa Veterinary Good performance in 1 lab in 2015 animal EQA

study in animals

ELISA Rift Valley Fever recN Ag detection ELISA Ruminants BDSL Veterinary Company no longer exists

IFA RVFV IIFT IgG. Human EUROIMMUN CE No published independent studies

IFA RVFV IIFT IgM Human EUROIMMUN CE No published independent studies

Lateral Flow Ingezim FVR Crom Ruminants Ingenasa Veterinary No published independent studies; does not

discriminate IgG vs. IgM

Dipstick VectorTest RVFV (antigen) Mosquito VectorTest - No published independent studies

ELISA

(in dev)

anti-RVFV IgM Humans ID-vet plan for CE Status unknown

Lateral flow

(in dev)

Device to detect IgM + IgG - USDA not known Status unknown




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ELISA IgG ELISA assay ID Screen Rift Valley Fever

Competition (IgM + IgG)

Multi-species ID-vet Veterinary Good performance in 9 labs in 2015 animal

EQA study and animal ring trial

ELISA ID Screen Rift Valley Fever IgM Capture Ruminants ID-vet Veterinary Good performance in 10 labs in 2015 animal

EQA study

ELISA Ingezim FVR DR 13-FVR.K0 (IgM) Ruminants Ingenasa Veterinary No published independent studies identified

ELISA Ingezim FVR Compac 13.FVR.K Ruminants Ingenasa Veterinary Good performance in 1 lab in 2015 animal EQA

study in animals

ELISA Rift Valley Fever recN Ag detection ELISA Ruminants BDSL Veterinary Company no longer exists

IFA RVFV IIFT IgG. Human EUROIMMUN CE No published independent studies

IFA RVFV IIFT IgM Human EUROIMMUN CE No published independent studies

Lateral Flow Ingezim FVR Crom Ruminants Ingenasa Veterinary No published independent studies; does not

discriminate IgG vs. IgM

Dipstick VectorTest RVFV (antigen) Mosquito VectorTest - No published independent studies

ELISA

(in dev)

anti-RVFV IgM Humans ID-vet plan for CE preliminary studies completed, validation

underway in several academic labs. No

timeline for launch

Lateral flow

(in dev)

Device to detect IgM + IgG - USDA not known Status unknown




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Status of lab-developed tests for Rift Valley Fever

Type Publication Description EQA status & Performance

RT-PCR Bird et al. (2007) 2 step real-time assay, 4 – 5 hours. Designed to detect 40

known strains. Designed for high throughput, automation.

8 labs used this protocol in 2012 human EQA study, where it showed

capacity for optimal performance. 2 labs used in 2015 animal EQA with

100% correct.

RT-PCR Drosten et al. (2002) 1 step real time assay. Quantitative. Part of a panel of assays

for 6 VHF pathogens

16 labs used this in 2012 human EQA study, showed capacity for optimal

performance. 3 labs used this in 2015 animal EQA study, 2 labs 100%, 1

lab had unacceptable results.

RT-PCR Drolet et al. (2012) 1 step real-time assay with pathogen inactivation in 1st

step. Study used samples with known status. No independently published

studies of performance.

LAMP Peyrefitte et al. (2008) 1 step endpoint assay. 63 C, 30 minute assay. Qualitative. Study used samples with known status, and artificial samples. No

independently published studies

LAMP Le Roux et al. (2009) 1 step Real time assay, 30 to 45 min assay 1 lab used this protocol in 2012 human EQA, where it showed limitations in

sensitivity.

RPA Eular et al. (2012) 1 step, 42 C, ESEquant tube scanner. < 30 min assay.

TwistDx reagents. Qualitative. Part of panel of 10 biothreat

pathogens.

1 lab used this protocol in 2012 human EQA, with optimal performance.

RT-PCR Weidmann et al. (2008) One step assay, designed against 19 strains 4 labs used this protocol in 2012 human EQA, showed capacity for optimal

performance.

RT-PCR Garcia et al. (2001) 2 step real time Taqman 1 lab used this in 2012 human EQA, did not detect Egypt strain.

RT-PCR Busquets et al. (2010) 1 step real time Taqman 1 lab used this in 2012 human EQA, did not show optimal performance.

RT-PCR Mwaengo et al. (2012) 2 step real time assay 1 lab used this in 2012 human EQA, did not show optimal performance.

RT-PCR Sall et al. (2002) Nested PCR assay, qualitative 6 labs used this in 2012 human EQA with mixed performance, some issue

with false positives.

RT-PCR Sanchez-Seco (2003) Nested PCR assay, qualitative 1 lab used this in 2012 human EQA, did not show optimal performance.

RT-PCR Wilson et al. (2014) Real time RT-PCR multiplex, detects 3 segments (different

dyes) allowing DIVA. Includes armored RNA ampli. control

Study used samples with known status. Low sensitivity reported for

quadriplex assay. No independent published studies

RT- PCR Liu et al. (2016) Taqman assay card for 26 pathogens, for outbreak

investigation or surveillance

Publication reports performance on > 1000 samples from various sources,

but small N for many rare pathogens




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Status of lab-developed tests for Rift Valley Fever

Type Publication Description EQA status & Performance

ELISA Paweska et al. (2003) Separate IgG sandwich and IgM capture assays for ruminants,

using irradiated whole virus as the antigen

IgG and IgM assays compared in van Vuren et al. (2010), against

samples with known status: good performance, IgM assay detected

seroconversion earlier (day 4).

ELISA Paweska et al. (2005) IgG sandwich and IgM capture assays for humans made using

irradiated whole virus as antigen, validated on human samples

Compared in van Vuren et al. (2010), against samples with known

status. Some problems with high viremia

ELISA Paweska et al. (2007) IgG assay for humans, made using recombinant N protein.

Validated in human samples

No independent EQA studies identified. Excellent performance in ~

3000 samples. Comparator was VNA

ELISA van Vuren et al. (2007) Separate IgG, IgM indirect ELISAs for humans, ruminants, uses

recombinant N protein.

Study used samples with known status. No independent EQA studies

identified.

ELISA Fafetine et al. (2007) Separate IgM and IgG assays for ruminants based on recombinant

N protein, validated in ruminants

Study used samples with known status. IgG assay compared in van

Vuren et al. (2010), less sensitive than IgG LDT 1 in detecting early

response in sheep

ELISA van Vuren et al. (2009) Sandwich ELISA for ruminants, humans. Uses polyclonal capture

sera generated using recombinant antigen. Does not dis-tinguish

IgM vs. IgG. Validated preincubation of samples at 56 C 1 hour

(biosafety).

Sensitivity poor compared to VNA. No independent EQA studies

identified

ELISA Jackel et al. (2013) Assay for ruminants uses recombinant Gn protein expressed in E.

coli, and detects IgG in sheep and goats

No independent EQA studies identified. Excellent performance in ~

2000 samples. Comparator was VNA

ELISA (DIVA) McElroy et al. (2009) 2 parallel ELISAs distinguish natural infections from vaccinations

(recombinant N and NSs proteins). Validated for goat, human.

Does not distinguish IgM vs IgG.

No independent EQA studies identified. Study used samples with

known infection status

ELISA Williams et al. (2011) IgM capture ELISA for ruminants, using rabbit anti-sheep IgM on

plate, and recombinant N protein conjugated to HRP for detection.

Validated on sheep

Some challenges in 2013 animal ring trial. Comparator was ID-vet

competition ELISA

ELISA Ellis et al. (2014) Non-species-specific assay, uses recombinant N protein expressed

in E. coli to coat plate, and N-protein-HRP conjugate for detection.

Detects both IgM and IgG. Validated on sheep and cattle.

No independent EQA studies identified. Comparator was ID-vet

ELISAs

VNA Winchger Schreur (2017) Avirulent RVFV which expresses eGFP, takes 48 hours and is

more sensitive than classic VNT. Not species specific.

No independent EQA studies identified. Comparator was classical VNA




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Recommendations for future planning

Diagnostic gaps for RVFV

– Validated, commercial serology assays for human specimens

– Diagnostics for lower-level laboratories

– Understanding of correlate(s) of protection

Lab capacity must be assessed and strengthened prior to beginning vaccine clinical trials and

epidemiological studies

Laboratory tests must be evaluated prior to starting clinical trials

– If appropriate tests are not available sufficient time must be allowed for R&D

– Access to well-characterized samples and reference standards can accelerate test evaluation

Procurement, import and export are additional considerations that can lead to unexpected delays in starting a

study

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Swiss Development and Coop eration;

State Secretariat for Education, Research and Innov ation

Netherlands EnterpriseAg ency

We also recognise the funding

partners who make the work of

FIND possible

FIND is a global non-profit driving diagnostic innovation ... · 8 labs used this protocol in 2012...

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